Enzymatic monohydrolysis of prostaglandin diester

ABSTRACT

The use of citrus acetyl esterase to selectively hydrolyze one of the ester functions present in 15-(R)-acetoxy-9-oxoprosta-5cis-10,13-trans-trienoic acid, methyl ester (15-epi-PGA2, acetate, methyl ester) is disclosed. The product of this selective hydrolysis is a useful intermediate for the synthesis of other physiologically active Prostaglandins.

United States. Patent [191 Leeming 1 Oct. 30, 1973 ENZYMATIC MONOHYDROLYSIS OF PROSTAGLANDIN DIESTER [75] Inventor: Michael R. G. Leeming, Canterbury,

England [73] Assignee: American Home Products Corporation, New York, N.Y.

221 Filed: Dec.28, 1971 21 Appl. No.: 213,187

[52] US. Cl. 195/51 R, 195/30 [51] Int. Cl Cl2d 1/02 [58] Field of Search 195/2 P, 30, 36 C,

[56] References Cited UNITED STATES PATENTS 3,687,811 8/1972 Colton et al. 195/30 FOREIGN PATENTS OR APPLICATIONS 111,971 8/1962 Pakistan OTHER PUBLICATIONS Jeffery et aL, Biochem J. Vol. 81, p. 591-596, 1961.

Primary Examiner-Alvin E. Tanenholt; Att0rney-Vito Victor Bellino et a1.

[57] ABSTRACT 5 Claims, No Drawings 1 ENZYMATIC MONOI-IYDROLYS IS OF PROSTAGLANDIN DIESTER BACKGROUND OF THE INVENTION A major source of PGE is isolation from mammalian tissue, a costly and inefficient means of obtaining this sought after material. It would thus be desirable to have another source for this material; this additional source would, ideally, be non-mammalian in origin.

The gorgonian coral Plexaura Homomalla (Esper) has been shown to contain a material which is useful as an intermediate for PGE methyl ester. Thus the 15 epimeric Prostaglandin A methyl ester (l5-epi-PGA methyl ester):

may be converted to PGE methyl ester by the method of G. Bundy, F. Lincoln, N. Nelson, J.'Pike, and W. Schneider, Annals. N. Y. Acad. Sci., 180, 76 (I971). In that procedure, the necessary intermediate, l5-epi- PGA methyl ester, is prepared by esterification of the carboxyl group of l5-epi-PGA which is isolated from Plexaura Homomalla (Esper) as a-minor product. The diester, l5-epi-PGA,, acetate, methyl ester which is isolated from Plex aura Homomalla (Esper) in amounts greater than 600% those of IS-eip-PGA, is not utilized in this sequence. It is thus the object of this invention to conveniently provide large quantities of IS-epi- PGA,, methyl ester, the required intermediate for the also known as IS-epi-Prostoglandin-A methyl ester,

or l5-epi-PGA methyl ester is provided. Examination of this compound produced according to the hereinafter described process reveals upon infrared, ultraviolet, proton magnetic, and mass spectrographic analysis, spectral data which fully supports the molecular structure as hereinbefore set forth.

The product of the process of the invention possesses the inherent use characteristic of being an intermediate in the synthesis of other Prostaglandin molecules, especially PGE methyl ester.

0Q-oH=cH-IH-cmcmcrncmon, H

DESCRIPTION OF THE PREFERRED EMBODIMENTS According to the process of the present invention, it has now been. found possible to selectively hydrolyze the ester function at the 15 position of 15-(R)-acetoxy- 9-oxoprosta-5-cis-l0,l 3-trans-trienoic acid methyl ester (l5-epi-PGA acetate, methyl ester) without concurrent hydrolysis of the methyl ester at the l position. This selective hydrolysis is accomplished by use of synthesis of PGE- methyl ester, from l5-epi-PGA acetate, methyl ester.

SUMMARY OF THE INVENTION The invention-sought to be patented resides in the concept of a process for preparing l5-(R)-hydroxy-9- citrus acetyl esterase, which may be prepared by the method of E. F. Jansen, R. Jang, and L. R. MacDonnell, Arch. Biochem., 15, 415 (1947). (The use of citrus acetyl esterase in cephalosporin chemistry has been According to the process of the present invention, the l 5-(R)-acetoxy-9-oxoprosta-5-cis-10,1 3-transtrienoic acid, methyl ester (l5-epi-PGA acetate, methyl ester) is contacted with citrus acetyl esterase in aqueous medium-for from about 5 to about 60 hours preferably atabout room temperature. The temperature is not critical, but should be kept below about 60 to avoid inactivation of the esterase. The pH of the aqueous medium may vary from about 5.5 to 7.5 but is ideally maintained at 6.5 throughout the reaction period. In addition, the l5-epi-PGA- acetate, methyl ester may be adsorbed on a finely divided, inert substrate such as Celite prior to its contact with the citrus acetyl esterase, or the l5-epi-PGA acetate, methyl ester, may be conveniently emulsified in an aqueous medium as for example by use of an ultrasonic vibrator prior to its contact with citrus acetyl esterase. It will be obvious to'one skilled in the art of organic chemistry,

The lS-epi-PGA methyl ester so obtained may then be converted to the pharmacologically useful compound, PCT-3 methyl ester according to the method described in Bundy et al, supra. I

The best mode contemplated by the inventor of carrying out his invention will now be set forth in the following non-limiting examples:

EXAMPLE 1 15-(R)Hydroxy-9-oxoprosta6-cis-10,13-transtrienoic acid, methyl ester (l-epi-PGA methyl ester) An emulsion of l5-(R)-acetoxy-9-oxopr0sta-5'cis- 10,1 B-trans-trienoic acid methyl ester (l5-epi-PGA acetate methyl ester) (100 mg.) in water (100 ml.) at 5C was prepared using an ultrasonic vibrator. This was treated with a solution of citrus acetyl esterase (20 ml.) prepared according to E. F. Jansen, R. Jang, and L. R. MacDonnell, Arch. Biochem. 15, 415 1947). The pH of the enzyme solution had previously been adjusted to 6.5. The reaction mixture was then shaken at 30C under nitrogen for 17 hours, allowed to stand at room temperature for 7 hours then shaken at30 for a further 17 hours. The mixture was then extracted with ether (5 X 50 ml.) and the extracts were washed with brine then dried over magnesium sulfate. Filtration and evaporation in vacuo gave a yellow oil (96 mg.).

EXAMPLE u l 5-(R)-Hydroxy-9 oxoprosta-5-cis-10, l 3-transtrienoic acid, methyl ester (IS-epi-PGA methyl ester) 1 5-(R)-Acetoxy-9-oxoprosta-5-cislO, l'3-transtrienoic acid, methyl ester (lS-epi-PGA: acetate methyl ester) (107 mg.) in ether (20 ml.) was treated with Celite (5 g.) and the solvent was removed in vacuo 20C. The resulting solid in water (100 ml.) was treated with citrus acetyl esterase solution which had previously been adjusted to pH 6.5. The mixture was shaken at 30C for 17 hours then was allowed. to stand at room temperature for 24 hours. It was then filtered and the filter cake was washed well with ether. The filtrate was then extracted with ether (5 X 50 ml.) and the combined filtrate and extracts were washed with brine and dried over magnesium sulfate. Filtration and evapacetoxy-Q-oxoprosta-S-cisl O ,l 3-trans-trienoic oration in vacuo gave an oil (76 mg.).

EXAMPLE Ill The products of examples I and ll were identical by TLC (silica gel, ethyl acetate-benzene) and they were therefore combined and chromatographed over silica gel (7g.), using a gradient of ethyl acetate in benzene. Two major fractions were obtained. l5-Epi-PGA acetate methyl ester (43 mg.) and a more polar compound (50 mg. This more polar compound was shown to be l5-(R)-hydroxy-9-oxoprosta-5-cis-10,13-transtrienoic acid, methyl ester (l5-epi-PGA methyl esterw 3470, 1745, 1710, 1590 cm. )\,,,,,,3216 mp. shifting to 278 mp. on base treatment. The NMR and mass spectra were also in full agreement with this structure.

The subject matter which the applicant regards as his invention is particularly pointed out and distinctly claimed as follows:

1. A process for preparing 15-(R)-hydroxy-9- oxoprosta-S-cis-IO,l3-trans-trienoic acid methyl ester which comprises contacting l5-'(R)-acetoxy-9- oxoprosta-S-cis-IO,l3-trans-trienoic acid methyl ester with citrus acetyl esterase.

2. The process as in claim 1 wherein the l5-(R)- acetoxy-9-oxoprosta-5-cis-10,1 3-trans-trienoic acid is contacted with citrus acetyl esterase in aqueous medium at a pH from 5.5 to 7.5.

3. The process as in claim 1 wherein the l5-(R)- acetoxy-9-oxoprosta-5-cis-10,l 3-trans-trienoic acid methyl ester is contacted with citrus acetyl esterase in aqueous medium at a pH of 6.5.

4. The process as in claim 1 wherein the l5-(R)- acetoxy-9-oxoprosta-5-cisl 0,1 B-trans-trienoic acid methyl ester is adsorbed on a finely divided inert substrate prior to its contact with citrus acetyl esterase.

5. The process as in claim 1 wherein the l5-(R)- acid methyl ester is emulsified in an aqueous medium prior to its contact with citrus acetyl esterase. a :k 4 

2. The process as in claim 1 wherein the 15-(R)-acetoxy-9-oxoprosta-5-cis-10,13-trans-trienoic acid is contacted with citrus acetyl esterase in aqueous medium at a pH from 5.5 to 7.5.
 3. The process as in claim 1 wherein the 15-(R)-acetoxy-9-oxoprosta-5-cis-10,13-trans-trienoic acid methyl ester is contacted with citrus acetyl esterase in aqueous medium at a pH of 6.5.
 4. The process as in claim 1 wherein the 15-(R)-acetoxy-9-oxoprosta-5-cis-10,13-trans-trienoic acid methyl ester is adsorbed on a finely divided inert substrate prior to its contact with citrus acetyl esterase.
 5. The process as in claim 1 wherein the 15-(R)-acetoxy-9-oxoprosta-5-cis-10,13-trans-trienoic acid methyl ester is emulsified in an aqueous medium prior to its contact with citrus acetyl esterase. 